Phage Display Using Cotranslational Translocation of Fusion Polypeptides

ABSTRACT

The present invention relates to a filamentous phage display method wherein the polypeptides of interest displayed on the phage particle are cotranslationally translocated across the cytoplasmic membrane of Gram-negative bacteria based on the signal recognition particle pathway. This method is particularly suitable for polypeptides, which are known to be difficult to display on phages, and for proteins of cDNA libraries and other combinatorial libraries, in particular when derived from very fast folding, stable protein scaffolds. The invention further relates to phage or phagemid vectors useful in the method comprising a gene construct coding for a fusion polypeptide comprising the polypeptide to be displayed on the phage particle and an N-terminal signal sequence promoting cotranslational translocation.

FIELD OF THE INVENTION

The present invention relates to a novel phage display method, phage orphagemid vectors used therein and the phage particles so obtained.

BACKGROUND OF THE INVENTION

Display of polypeptides on bacteriophage (phage display) is a selectiontechnique that allows to extract polypeptides with desired propertiesfrom a large collection of variants (Russel, M., Lowman, H. B., andClackson, T., Introduction to phage biology and phage display, in “PhageDisplay”; Clackson, T. and Lowman, H. B., eds., Oxford University Press,2004, pp. 1-26). Phage display has been intensively investigated for theselection from combinatorial antibody or peptide libraries.

By far the most widely used bacteriophages used in phage display arefilamentous phages. Filamentous phages constitute a large family ofbacterial viruses that infect many Gram-negative bacteria. Thebest-known filamentous phages are those that infect Escherichia coli;these are f1/M13/fd and IKe. Phages f1, M13, and fd are those that haveso far been used for filamentous phage display. Their genomes are morethan 98% identical and their gene products are interchangeable.

A unique aspect of filamentous phage assembly, in contrast to theassembly of many other bacteriophages, is that it is a secretoryprocess. Incorporation of coat polypeptides into the growing phageoccurs in the cytoplasmic membrane, and nascent phages are extruded fromthe cell as they assemble (Russel et al., loc. cit.). The E. coli celldoes not lyse in this process. The five viral coat proteins (pIII, pVI,pVII, pVIII and pIX) are inserted in the cytoplasmic membrane prior totheir incorporation into phage particles (FIG. 1). For example, themajor part of pIII is translocated across the membrane into theperiplasm, while its C-terminal hydrophobic tail anchors the protein inthe membrane.

One prerequisite for filamentous phage display is the translocation ofthe polypeptide of interest (POI) across the cytoplasmic membrane. Thisis normally achieved by genetically fusing the POI to a phage coatprotein and translocation of the corresponding fusion polypeptide.Alternatively, the POI and phage coat protein are translocatedindependently.

In this situation the POI is stably linked to the phage particle in theperiplasm by, for example, formation of a disulfide bond (Cys-Display)or formation of a leucine-zipper (pJuFo system) with a correspondingphage coat protein. In conventional filamentous phage display usingfusions to pIII, the Sec pathway is used for translocation of the fusionpolypeptide comprising the POI. In this pathway, the polypeptide isfirst synthesized at the ribosome and then posttranslationallytranslocated, in its unfolded state, by the Sec translocon (FIG. 2,(3)-(4)-(5)). That is, the translocation across the cytoplasmic membranebegins only after a substantial amount of the polypeptide chain has beensynthesized. However, the contribution of the mechanism of translocationfor the success of phage display has not been fully elucidated, and thepossibility to use a cotranslational translocation pathway was notexplored in the prior art.

Intracellular and extracellular proteins of a wide range of sizes andstructures have been functionally displayed on filamentous phage (Russelet al., loc. cit.). Nevertheless, some polypeptides are recalcitrant todisplay due to individual properties, mostly because of unknown reasons.This makes the success of the display of a certain proteinunpredictable. Thus, it has usually been recommended to first test theefficiency of display on filamentous phage for each protein to be used.In addition, when a combinatorial library is created for phage display,not all clones will display with similar efficiency; this is especiallytrue for libraries generated from cDNAs. The display problems ofpolypeptides may be a result of their interference with the phageproduction, their periplasmic aggregation, their proteolysis, theirtoxicity to E. coli or their incompatibility with the used translocationpathway. Especially, the step preceding translocation is an importantfactor influencing the incorporation of the fusion polypeptides into thephage particles. If the polypeptides fold prematurely, they can berefractory to translocation or even exhibit cytoplasmic toxicity. Thus,it is important whether the protein is translocated posttranslationally(potentially allowing premature folding) or cotranslationally (notpermitting cytoplasmic folding). Current filamentous phage displaymethods use posttranslational pathways for translocation of the fusionpolypeptide across the cytoplasmic membrane (Russel et al., loc. cit.;Paschke, M. and Höhne, W., Gene 350, 79-88, 2005). Thus, polypeptidesincompatible with these pathways will be refractory to display, makingphage display selections very inefficient or even impossible. Forexample, the posttranslational Sec pathway, which is almost exclusivelyused in phage display, is inherently incapable of translocating proteinsthat cannot remain in an unfolded state in the cytoplasm, since the Sectranslocon itself can only transport unfolded polypeptides (Huber, D.,Boyd, D., Xia, Y., Olma, M. H., Gerstein, M., and Beckwith, J., J.Bacteriol. 187, 2983-2991, 2005; Paschke et al., loc. cit.).

Thus, the technical problem underlying the present invention is toidentify novel translocation approaches for the efficient display ofthose polypeptides on filamentous phages that are displayedinefficiently by using posttranslational translocation. The solution tothis technical problem is achieved by providing the embodimentscharacterized in the claims.

SUMMARY OF THE INVENTION

The present invention relates to a filamentous phage display methodwherein the polypeptides of interest (POI) displayed on the phageparticles are cotranslationally translocated across the cytoplasmicmembrane of Gram-negative bacteria, in particular based on the signalrecognition particle pathway.

Accordingly, the present invention allows phage display bycotranslational translocation of the fusion polypeptides comprising thePOI. This method is particularly suitable for polypeptides, which areknown to be difficult to display on phages, and for proteins of cDNAlibraries and other combinatorial libraries, in particular when derivedfrom very fast folding, stable protein scaffolds.

The invention further relates to phage or phagemid vectors comprising agene construct coding for a fusion polypeptide comprising the POI to bedisplayed on the phage particle and an N-terminal signal sequencepromoting cotranslational translocation based on the signal recognitionparticle pathway, and to phages obtained by the method of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Membrane Insertion and Display of the Polypeptide of Interest

N-termini (N), C-termini (C), polypeptide of interest (POI); N-terminaldomains of pIII (N1, N2), C-terminal domain of pIII (CT).

A) The display of the polypeptide of interest (POI) on a filamentousphage particle always includes translocation of the POI across thecytoplasmic membrane (cm) into the periplasm (pp). Most often, the POIis translocated as a fusion polypeptide including the coat protein III(pIII) or a fragment thereof. pIII comprises two N-terminal domains (N1,N2) and the C-terminal domain (CT). In this specific example, the fusionpolypeptide consists of the N-terminal POI and the C-terminal CT. Thefusion polypeptide and pIII are anchored to the cytoplasmic membranethrough a C-terminal hydrophobic stretch at the CT moiety aftertranslocation and prior to their incorporation into the phage particle.Cytoplasm (cp), outer membrane (om), extracellular space (ex).

B) A simplified view of a filamentous phage particle displaying pIII andthe fusion polypeptide of A). The N-terminal domains of pIII (N1, N2)and the POI are incorporated into the phage particle via the CT moiety.

FIG. 2. Translocation of Polypeptides Across the Cytoplasmic Membrane ofGram-Negative Bacteria.

A simplified view of the three major pathways known for thetranslocation of polypeptides across the cytoplasmic membrane (cm) intothe periplasm (pp) of Gram-negative bacteria. These pathways are the SRPpathway, the Sec pathway and the Tat pathway. Both the Sec and the SRPpathway rely on the Sec translocon (SecTR), whereas the Tat pathwayrelies on the Tat translocon (TatTR). Both the Sec and the Tat pathwaytranslocate polypeptides posttranslationally whereas the SRP pathwaytranslocates polypeptides cotranslationally. The Sec and SRP pathwaysconverge at the Sec translocase that transports the proteins in anunfolded (uf) state through the membrane. In contrast, the Tattranslocon only translocates folded (f) proteins. The amino acidcomposition of the signal sequence (ss) strongly favors the targeting ofthe preprotein to one of these pathways. After translocation, the signalsequence is cleaved off from the preprotein by a peptidase. The SRPpathway is mediated by the signal recognition particle (SRP), aribonucleoprotein consisting of the 54-kDa protein homolog and a 4.5SRNA. In this pathway, it is the SRP that targets the preprotein to theSec translocon. The SRP recognizes and binds corresponding signalsequences emerging from the ribosome (R), delivers the ribosome-nascentchain complex to the SRP-receptor (SR) and subsequently, the preproteinis cotranslationally translocated through the Sec translocon.Polypeptides that are incompatible with the Sec pathway because of theirpremature cytoplasmic folding can thus be efficiently translocated bythe SRP pathway while being translated. (1) The SRP binds toparticularly hydrophobic signal sequences of nascent proteins emergingfrom the ribosome. (2) The SRP directs the ribosome nascent chaincomplex via the SRP-receptor to the Sec translocon, were thecotranslational translocation takes place. Most preproteins have lesshydrophobic signal sequences and undergo SecB dependent export. (3)Trigger factor (TF), a cytosolic chaperone that has a general affinityfor nascent polypeptides, binds to the mature region of nascentpreproteins and remains effectively bound until the translation isalmost finished. (4) Following TF dissociation, cytosolic factors suchas SecB help to maintain preproteins in an extended unfoldedconformation. (5) Preproteins that retain an extended conformation areefficiently transported trough the Sec translocon. (6) However, iffolding of the preprotein occurs in the cytoplasm, the protein isusually degraded (10) or may even plug up the Sec translocon.

Preproteins with signal sequences containing the twin-arginine motif aredestined to the Tat translocon. (7) Association with a chaperone (TC),such as DnaK or another Tat-specific factor, probably shields the signalsequence until folding is completed (8). This same factor or anadditional factor may also promote correct folding. Tat translocationproceeds only if the preprotein is correctly folded; otherwise, thepreprotein is degraded by the proteolytic machinery (9, 10) of the cell.

FIG. 3. Schematic Representation of the pDST Phagemid Vector Series.

A) Enlarged view of the expression cassette of the pDST phagemid vectorseries. The expression cassette comprises a promoter/operator element ofthe lacZgene of E. coli (lacZ p/o), a ribosome binding site (notdepicted), the coding sequences for the signal sequence (ss) and apolypeptide of interest (POI) to be displayed, a suppressor stop codon(TAG), the coding sequences fora flexible glycine/serine linker (G/S)and for the C-terminal domain (amino acids 250-406) of protein III offilamentous phage (CTpIII) mediating incorporation of the fusionpolypeptide into the phage particle, two stop codons (TGATAA, notdepicted) and a transcription terminator element (not depicted). Thecoding sequence of the POI is flanked by DNA sequences encoding aFlag-tag (Flag) and a c-myc-tag (c-myc). The single letter amino acidsequences for the DsbA signal sequence (DsbAss) as a representative ofsignal sequences targeting the SRP pathway and for the PhoA signalsequence (PhoAss) as a representative of a signal sequences targetingthe Sec pathway are shown. These signal sequences contain a positivelycharged N-terminal region (n-region), an apolar hydrophobic core(h-region) and a more polar C-terminal region (c-region).

B) Schematic representation of the phagemid pDST23. In addition to theelements shown in A) the filamentous phage replication origin (Ff ori),the lac repressor gene from E. coli (lacI), which produces lac repressorneeded for the tight control of the lacZ p/o, the ColE1 origin ofreplication (ColE1 ori) for bacterial replication of the vector, theantibiotic resistance gene (cat) encoding achloramphenicol-acetyl-transferase mediating chloramphenicol resistanceand the restriction sites XbaI, BamHI, PstI, and EcoRI are depicted. Thein pDST23 encoded POI is the designed ankyrin repeat protein (DARPin)3a.

FIG. 4. Display Yield Comparison by Western Blot Analysis

A) and B) CsCl-purified phage particles produced by the use of therespective phagemids were separated by SDS-PAGE, blotted onto PVDFmembranes and detected with antibodies specific for the C-terminaldomain of protein III (anti-pIII) or the Flag-tag (anti-FlagM1).Aliquots applied per lane have been normalized and correspond to 5×10¹¹phage particles. The display yields on phage particles for variouspolypeptides are analyzed. The abbreviated names of the polypeptides areindicated on top of the lanes and refer to the polypeptides listed inTable 1. In addition, Table 1 indicates the corresponding phagemids usedto produce the phage particles, and an outline of the expressioncassette is given in FIG. 3A. The display yields are compared for eachpolypeptide using either the PhoA signal sequences (lanes labeled “p”)or the DsbA signal sequence (lanes labeled “d”) to translocate thecorresponding fusion polypeptide by the Sec pathway or the SRP pathway,respectively. Stronger bands indicate higher display yields. Themolecular weights of marker proteins in kDa are indicated at both sidesof the blot. The band at 62 kDa in the anti-pIII blot corresponds to thepIII wild-type protein.

FIG. 5. Display Yield Comparison by ELISA Analysis

Phage particles displaying either the cJun N-terminal kinase 2(JNK2)-binding. DARPin called 2_(—)3 (open symbols) or theaminoglycoside kinase APH(3′)-IIIa (APH)-binding DARPin called 3a(filled symbols) were incubated in neutravidin coated and BSA-blockedwells containing immobilized biotinylated JNK2 or APH proteins,respectively. After washing, the bound phage particles were detectedwith anti-M13 antibody coupled to horseradish peroxidase and visualizedwith soluble BM Blue POD substrate. The data plotted show the absorbance(Abs) on the y-coordinate measured at 360 nm after subtracting thebackground measured at 392 nm versus the number of phage particles (pp)applied per well on the x-axis. Phage particles produced using the DsbAsignal sequence encoding phagemid variants (triangle symbols) showedhalf-maximum signal already at about 8×10⁸ phagesper well, whereas phageparticles produced from PhoA signal sequence encoding variants (squaresymbols) showed half-maximum signal only at about 2×10¹¹ phage particlesper well, indicating more than 100-fold lower display yields. Thus, theuse of DsbA signal sequence-mediated SRP pathway for the translocationof the fusion polypeptides strongly increased the display yields incomparison to using the PhoA signal sequence-mediated Sec pathway.

FIG. 6. Quantification of Display Yield by ELISA Analysis

Phage particles displaying DARPin 2_(—)3 or 3a were analyzed asdescribed for FIG. 5. The results are given in a column diagram(logarithmic scale) with PhoAss display yields set to 1. The displaylevel (fold increase) for each signal sequence used corresponds to thenumber of phage particles giving a signal of OD₄₅₀=0.5 relative to thenumber of PhoAss-containing phage particles giving a signal ofOD₄₅₀=0.5. PelBss: Signal sequence of Erwinia carotovora PeIB (aputative Sec-dependent signal sequence). SRP-dependent signal sequencesof the E. coli proteins TorT (TorTss), TolB (TolBss) and SfmC (SfmCss)were tested in addition to the DsbAss. An increased display yield of upto 700-fold was observed with the SRP-dependent TorTss compared to theSec-dependent PhoAss. The SRP-dependent TolBss and SfmCss gave anincreased display yield up to 300-fold. The putative Sec-dependentPeIBss showed only a two- to sixfold increased display yield.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the present invention, the term “filamentous phagedisplay” refers to phage display based on filamentous phages.Filamentous phages constitute a large family of bacterial viruses thatinfect many Gram-negative bacteria. Preferred filamentous phages arethose that infect E. coli; in particular f1/M13/fd and IKe. Methods topractice filamentous phage display are well known to the person skilledin the art (e.g. Russel et al., loc. cit.).

In the context of the present invention, the term “signal sequence”refers to an N-terminal stretch of amino acids of a polypeptideresulting in targeting of the polypeptide to a translocase. In E. coli,N-terminal signal sequences generally comprise 15 to 52 amino acids.Most signal sequences contain a positively charged N-terminal region(n-region), an apolar hydrophobic core (h-region) and a more polarC-terminal region (c-region). The c-region contains the cleavage sitefor signal peptidase. Signal peptidase is a membrane-bound protease thatremoves the signal sequence from the polypeptide during thetranslocation reaction. Such signal sequences comprising 18 to 30 aminoacids are preferred. The determination of signal sequences is well knownto the person skilled in the art. For example, they can be obtained fromdatabases such as Swiss-Prot or GenBank or using annotated genome-widedata sets.

In the context of the present invention, the term “preprotein” refers toa polypeptide comprising an N-terminal signal sequence. The signalsequence is cleaved from the preprotein during the translocationreaction thus yielding the mature protein.

In the context of the present invention, the term “fusion polypeptide”refers to a polypeptide comprising an N-terminal signal sequence, thepolypeptide of interest (POI) and an additional amino acid sequenceallowing display on filamentous phage. Preferably, this additionalsequence comprises a filamentous phage coat polypeptide or a fragmentthereof. Alternatively, this sequence connects the POI to the phageparticle in the periplasm by formation of a stable linkage, for example,by formation of a disulfide bond (Cys-Display) or by formation of aleucine-zipper (pJuFo system) with a corresponding phage coat protein.In this alternative strategy, the POI and the corresponding phage coatprotein are translocated independently across the cytoplasmic membrane.

In the context of the present invention, the term “translocation” refersto the translocation of a polypeptide across a biological membranemediated by a translocon (Holland, I. B. et al., Biochim. Biophys. Acta1694, 5-16, 2004). The translocation occurs posttranslationally orcotranslationally. A translocase is hence an enzyme or enzyme complexthat specifically transports a polypeptide through the translocon(Holland et al., loc. cit.).

In the context of the present invention, the term “Sec pathway” refersto a protein transport mechanism for posttranslational translocation ofpreproteins across the cytoplasmic membrane of Gram-negative bacteriathrough the Sec translocon (Holland et al., loc. cit.). The Sec pathwayis mediated by molecular chaperones, most often SecB, that keeppreproteins in an unfolded state before translocation. The Sec pathwayis the major route of protein translocation in Gram-negative bacteria.

In the context of the present invention, the term “SRP pathway” refersto a protein transport mechanism for cotranslational translocation ofpreproteins across the cytoplasmic membrane of Gram-negative bacteriathrough the Sec translocon (Schierle, C. F., Berkmen, M., Huber, D.,Kumamoto, C.; Boyd, D., and Beckwith, J., J. Bacteriol. 185, 5706-5713,2003; Huber et al., loc. cit.). The SRP pathway is mediated by thesignal recognition particle (SRP), a ribonucleoprotein consisting of the54-kDa protein homolog (Fifty-four homolog; Ffh) and a 4.5S RNA. In theSRP pathway, the signal sequence interacts with SRP as soon as itappears from the ribosome. The complex consisting of SRP, nascentpolypeptide and ribosome is then transferred via the SRP-receptor to theSec translocon where the polypeptide is cotranslationally translocatedthrough the Sec translocon.

The Sec and SRP pathways converge at the Sec translocase that transportsthe proteins in an unfolded state through the membrane. It is the aminoacid composition of the signal sequence that will strongly favor thetargeting of the preprotein to the SRP pathway over the Sec pathway forits translocation (Huber et al., loc. cit.).

In the context of the present invention, the term “DsbA” refers to theperiplasmic E. coli thiol:disulfide interchange protein DsbA (Swiss-Protaccession number P24991). DsbA is a substrate of the SRP pathway (Huberet al., loc. cit.). DsbA is exported cotranslationally to avoid itsfolding in the cytoplasm, which would inhibit its export.

In the context of the present invention, the term “TrxA” refers to theE. coli protein thioredoxin 1 (Swiss-Prot accession number P00274). TrxAcan be used as a reporter protein to distinguish signal sequences thattarget a preprotein to the SRP pathway or the Sec pathway (Schierle etal., loc. cit.; Huber et al., loc. cit.).

In the context of the present invention, the term “Tat pathway” refersto the twin-arginine protein translocation (Tat) pathway (Paschke etal., loc. cit.). The Tat pathway differs fundamentally from the Secpathway and SRP pathway. In contrast to the Sec translocon, the Tattranslocon exports only fully folded proteins. In contrast to the SRPpathway, the Tat translocon passes proteins posttranslationally throughthe membrane.

In one particular embodiment the signal sequence of the fusionpolypeptide comprising the POI to be displayed on the phage particle isa signal sequence promoting cotranslational translocation.

Methods to test if a signal sequence of interest promotescotranslational translocation across the cytoplasmic membrane ofGram-negative bacteria are well known to the person skilled in the art.For example, the signal sequence of interest is genetically fused to themature MaIE (Swiss-Prot accession number P02928, residues 27 to 396).This artificial preprotein is then expressed in E. coli and the yield ofcotranslational proteolytic processing of its nascent chain is analyzedby two-dimensional gel electrophoresis as, described (Josefsson, L.-G.and Randall, L. L., Methods Enzymol. 97, 77-85, 1983; Schierle et al.,loc.cit.). Removal of the N-terminal signal sequence of interest whilethe preprotein chains are still nascent indicates that translocation isinitiated before the synthesis of the polypeptide is complete and thusthat translocation is cotranslational. Preferred signal sequences arethose that promote yields of cotranslational translocation of over 80%,more preferably over 90%, when fused to mature MaIE. Most preferredsignal sequences are those that do only promote cotranslationaltranslocation and no posttranslational translocation when fused tomature MaIE. Alternatively, signal sequences already known to promotecotranslational translocation, such as the signal sequence from DsbA,may be used.

In another particular embodiment, the signal sequence of the fusionpolypeptide comprising the POI to be displayed on the phage particle isa signal sequence targeting the signal recognition pathway.

The signal recognition pathway of Gram-negative bacteria and methods totest if a signal sequence of interest targets a preprotein to the SRPpathway are well known to the person skilled in the art. For example,the translocation of TrxA fused to a signal sequence targeting the SRPpathway is strongly inhibited in E. coli bearing a mutation in the geneffh (e.g. a ffh77 or ffh87 mutant strain), which encodes a component ofthe SRP (Schierle et al., loc. cit.; Huber et al., loc. cit.). Thus,those signal sequences that target the SRP pathway promote translocationof TrxA across the cytoplasmic membrane in wild-type E. coli, but veryinefficiently in an ffh mutant strain. Signal sequences can thus begrouped into two distinct classes: Those that target the SRP pathway andthose that do not target the SRP pathway. Signal sequences that targetthe Sec pathway can be redirected to the SRP pathway by increasingoverall hydrophobicity of the signal sequence, in particular byincreasing the hydrophobicity of its h-region. For example, modestalterations of the MaIE signal sequence that simply increase itshydrophobicity by replacing polar or small (Gly or Ala) amino acids inthe h-region by large hydrophobic residues reroute the protein from theSec to the SRP pathway. Alternatively, signal sequences already known totarget the SRP pathway, such as the signal sequence from DsbA, may beused. Other examples of signal sequences using the SRP pathway are thoseof a subset of autotransporters, such as that of the hemoglobin protease(Hbp, UniProtKB accession number O88093). Unusually, the Hbp signalsequence is relatively long (52 amino acids) and contains a N-terminalextension that precedes a classical signal sequence. In addition, theh-region of the Hbp signal sequence is not particularly hydrophobic. Thesignal sequence of SecM (Swiss-Prot accession number P62395) is anotherexample of a long signal sequence that comprises an N-terminal extensionand a moderately hydrophobic h-region that is known to target the SRPpathway.

In still another particular embodiment, the signal sequence of thefusion polypeptide comprising the POI to be displayed on the phageparticle is a signal sequence promoting translocation of TrxA across thecytoplasmic membrane of Gram-negative bacteria.

Many commonly used signal sequences, e.g. those of PhoA (Swiss-Protaccession number P00634) and MaIE (Swiss-Prot accession number P02928),do only inefficiently promote the translocation of TrxA across thecytoplasmic membrane (Schierle et al., loc. cit.). In contrast, thesignal sequence from DsbA promotes efficient translocation of TrxA.Subcellular fractionation of host cells expressing TrxA fused to thesignal sequence of interest allows to discriminate those signalsequences that are able to promote translocation of TrxA across thecytoplasmic membrane into the periplasm from those that are not (Huberet al., loc. cit.). The quantity of TrxA in the periplasmic fractiondescribes the efficiency of the signal sequence to promote translocationof TrxA. Alternatively, signal sequences already known to promotetranslocation of TrxA, such as the signal sequence from DsbA, may beused.

In a preferred embodiment, the signal sequence of the fusion polypeptidecomprising the POI to be displayed on the phage particle is a signalsequence selected from the group consisting of TorT, SfmC, FocC, CcmH,YraI, TolB, NikA, FlgI and DsbA, and homologs thereof.

In a particularly preferred embodiment, the signal sequence of thefusion polypeptide comprising the POI to be displayed on the phage,particle is a signal sequence selected from the group consisting ofTorT, SfmC, TolB and DsbA.

In the context of the present invention, the term “homolog” of a signalsequence means an amino acid sequence with 70%, preferably 80%, and inparticular 90% or more amino acid identity with any of the signalsequences mentioned hereinbefore, while conserving the overall charge,hydrophobicity and cleavage properties of the n-region, h-region andc-region of the signal sequence, respectively. Examples of such homologsare amino acid sequences wherein one, two, three or four, in particularone or two, amino acids are replaced by other amino acids, wherein one,two, three or four amino acids are deleted, or one or two amino acidsare added, or combinations of replacements, deletions and additions asmentioned hereinbefore. In replacements of amino acids, apolar aminoacids are preferably replaced by other apolar amino acids, e.g. Ile byLeu, Val, Ala, Trp, Phe or Met or vice versa, polar amino acids by otherpolar amino acids, e.g. Thr by Ser, Asn or Gln or vice versa, negativecharged amino acids by other negative charged amino acids, e.g. Asp byGlu or vice versa, or positive charged amino acids by other positivecharged amino acids, e.g. Lys by Arg or His or vice versa

For example, for the preferred signal sequence of DsbA with the aminosequence MKKIWLALAG LVLAFSASA (SEQ ID NO:1), the replacement of aminoacid Lys2 by Arg, Ala9 by Leu, Ala14 by Val and Ser16 by Thr ispossible.

The signal sequences of TorT, SfmC, FocC, CcmH, YraI, TolB, NikA, FlgIand DsbA are known to promote cotranslational translocation of TrxA bytargeting the SRP pathway, and possess, in most cases, a higher overallhydrophobicity compared to signal sequences targeting the Sec pathway(Huber et al., loc. cit.). The proteins have the following. Swiss-Protaccession numbers: TorT P38683, SfmC P77249, FocC P62609, CcmH P33925,YraI P42914, TolB P0A855, NikA P33590, FlgI P0A6S3, and DsbA P24991.

Hydrophobicity calculations alone do not allow to discriminate SRPdependent and non-SRP dependent signal sequences in the highhydrophobicity range (Huber et al., loc. cit.). Thus, features otherthan hydrophobicity (e.g. the structure of the signal peptide) influencethe preference for one translocation pathway.

In a particularly preferred embodiment, the signal sequence is the DsbAsignal sequence or any amino acid sequence possessing 90% identity withthe DsbA signal sequence.

The method is applicable to any of the filamentous phage displaymethods, for example with phages f1, M13, fd and Ike, in particular f1,M13 and fd.

In particular, the method of the invention comprises the steps of

(a) constructing a filamentous phage or phagemid vector containing anexpression cassette for a fusion polypeptide that possesses anN-terminal signal sequence promoting cotranslational translocation ofthe fusion polypeptide across the cytoplasmic membrane of Gram-negativebacteria;

(b) constructing a combinatorial library of phage or phagemid vectors bycloning of a DNA library encoding the polypeptides of interests into theexpression cassette of the vector of step (a);

(c) transforming suitable Gram-negative bacteria with the library ofvectors of step (b); and

(d) performing phage display selection cycles to separate phageparticles based on the properties of the displayed proteins of interest.

In step (a), the filamentous phage or phagemid vector is constructedusing standard methods of gene technology. For example, the signalsequence encoding part of the expression cassette for the fusionpolypeptide of an established phage or phagemid vector is replaced bythe coding sequence of said signal sequence using standard DNAtechniques. Alternatively, a novel phage or phagemid vector containingan expression cassette for the fusion polypeptide containing said signalsequence is constructed de novo using general knowledge on thecomposition of such vectors (e.g. Russel et al., loc. cit) and standardDNA synthesis and assembly methods. Phage or phagemid vectors useful forthat purpose are, for example, pAK100, pComb3, pEXmide3, pHEN1, pJuFo orpSEX. An example of such a phagemid vector (pDST23) is described in FIG.3 and in the accompanying Example, as an illustration of the inventionwithout limiting the invention to this-particular embodiment.

Preferably, the signal sequence of the fusion polypeptide in step (a)promotes translocation of the fusion polypeptide through the SRPpathway.

More preferably, the signal sequence of the fusion polypeptide in step(a) promotes the cotranslational translocation of TrxA.

In step (b), standard methods for the preparation of combinatoriallibraries of vectors are used. For example, combinatorial DNA librariesencoding the proteins of interest are generated by random orsite-directed mutagenesis, by DNA shuffling, by preparation of cDNAthrough amplification of cellular mRNA or by consensus design and thenligated into the expression cassette of said vector by standard DNAtechniques.

In step (c), standard methods for the transformation of Gram-negativebacteria are used. For example, the bacteria are transformed by thecombinatorial library of vectors of step (b) by electroporation orchemical means. Such methods are well known to the person skilled in theart.

In step (d), standard phage display selection cycles are performed. Suchphage display selection cycles are well known to the person skilled inthe art (e.g. Russel et al., loc. cit.).

Preferably, the property of the displayed polypeptide of interest ofstep (d) is specific binding to a target molecule of interest. In thiscase, phage particles displaying a POI binding to the target moleculeare separated from phage particles displaying irrelevant polypeptides byapplying the amplified phage particles in each selection cycle to thetarget molecule functionally immobilized on a surface, washing unboundphage particles away, eluting the bound phage particles and using theeluted phage particles as input for the amplification of phage particlesof the next selection cycle.

It is understood that, whenever in the context of this invention, “asignal sequence” or “the signal sequence” is mentioned, such anexpression also means “one or more”, e.g. one, two, three or four,signal sequences of different composition. Using more than one signalsequences may be advantageous for particular applications, and is alsowithin the ambit of this invention.

The invention further relates to a phage or phagemid vector comprising agene construct coding for a fusion polypeptide comprising the POI fusedto an N-terminal signal sequence promoting cotranslational translocationof TrxA, in particular a signal sequence that is selected from the groupconsisting of signal sequences from TorT, SfmC, FocC, CcmH, YraI, TolB,NikA FlgI, and DsbA, and homologs thereof, preferably selected fromTorT, SfmC, TolB and DsbA. Most preferred is a phage or phagemid vectorcomprising the signal sequence DsbA or a homolog thereof.

Preferably, the fusion polypeptide comprises the POI fused to the phagecoat protein pIII or pVIII, or to a fragment of the coat protein pIII.Such a fragment is, for example, a fragment comprising amino acids 250to 406 of pIII.

The signal sequence of the periplasmic enzyme DsbA directs fusedreporter proteins to the SRP pathway and thus enhances theircotranslational export. This indicates that the relatively hydrophobicDsbA signal peptide interacts with SRP and promotes cotranslationaltranslocation of DsbA. Thus, the signal sequence of DsbA can be used asa generic signal sequence for other proteins, as will be shown below inthe Example.

Likewise, homologs of the signal sequence of DsbA, and signal sequencesof TorT, SfmC, FocC, CcmH, YraI, TolB, NikA, and FlgI and homologsthereof may be used.

The method of the invention is particularly suitable for the applicationwith libraries of compounds, for example DNA libraries, in particularcDNA libraries. In contrast to methods used hitherto in phage display,the method of the invention allows reliable presentation of polypeptidesobtained by expression of such libraries.

A particular embodiment of the invention is the described filamentousphage display method wherein POIs encoded by a DNA library are displayedon the phage particles. Preferably, cotranslational translocation forthe POIs encoded by a DNA library is accomplished based on the signalrecognition particle pathway, such as a signal sequence promotingcotranslational translocation of TrxA. Most preferred is the method asdescribed herein for the phage display of repeat proteins.

As with any selection technology, the success of phage displayselections strongly depends on the diversity of displayed librarymembers. A large combinatorial DNA library does not by itself guaranteethat a large diversity of library members can be displayed. In phagedisplay, the polypeptides to be displayed have to be translocated acrossthe cytoplasmic membrane before their incorporation into phageparticles. Current filamentous phage display methods all use aposttranslational pathway for translocation of the fusion polypeptideacross the cytoplasmic membrane (Russel et al., loc. cit.; Paschke etal., loc. cit.). Thus, library members incompatible with these pathwayswill be refractory to display thus clearly reducing the displayedlibrary diversity.

DNA libraries considered are all possible combinatorial DNA librariesincluding those produced by random or site-directed mutagenesis, by DNAshuffling, or by consensus design. Such methods will generate librarymembers with novel properties, such as binding properties; some of themwill be incompatible with the translocation using the Sec pathway. Suchlibraries include DNA libraries that encode peptide libraries, antibodylibraries or libraries based on alternative scaffolds (Russel et al.,loc. cit.; Nygren, P. A. and Skerra, A., J. Immunol. Methods, 290, 3-28,2004).

Further DNA libraries considered are cDNA libraries, especially those ofeukaryotic origin. cDNA libraries encode a great variety of naturallyoccurring cellular proteins including cytoplasmic proteins, membraneproteins and extracellular proteins. Some of these naturally occurringlibrary members will be incompatible with translocation using the Secpathway.

Further DNA libraries considered are those encoding single-chain Fv(scFv) antibody libraries that are used to select intracellularly activescFv fragments (intrabodies). A prerequisite for intrabodies is thatthey fold well and are stable in the cytoplasm of E. coli. Thus,cytoplasmic-stable and well-folded intrabodies are inefficientlytranslocated by the posttranslational mechanism of the Sec pathway.

Further DNA libraries considered are those encoding alternative scaffoldlibraries based on repeat proteins, including ankyrin repeat proteins,leucine-rich repeat proteins, tetratricopeptide repeat protein,pentatricopeptide repeat proteins or armadillo/HEAT repeat proteins.

A preferred DNA library is a library encoding designed ankyrin repeatproteins (DARPins) (Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T.,Briand, C., Forrer, P., Grütter, M. G., and Plückthun, A., Nat.Biotechnol., 22, 575-582, 2004). Examples of DARPins displayed on phageparticles are shown in the Example.

The invention further relates to the phages produced by the method ofthe invention.

The invention further relates to the periplasmic expression of very fastfolding and cytoplasmically stable proteins, in particular to theperiplasmic expression of DARPins. The DsbAss and DARPins encodingphagemids of the Examples can directly be used for the efficientperiplasmic expression of the DARPins in a non-suppressing E. coli.Alternatively, standard periplasmic expression vectors can be adapted byreplacing the DNA encoding the signal sequence used for periplasmicexpression by DNA encoding a signal sequence of the current invention,in particular by the DNA encoding the DsbAss. For example, the efficientperiplasmic expression of DARPins is instrumental to the expression ofDARPins fused to effector proteins, in particular toxins or cytokines,which are very difficult to express in the cytoplasm.

The new phage display method exemplified herein below allows toefficiently incorporate a very broad range of POIs to be displayed intophage particles and thus enables efficient phage display. The keydifference of this new method compared to traditional phage displaymethods is the use of signal sequences directing the fusion polypeptidecomprising the POI to be displayed to the cotranslational SRP pathway(FIG. 2, (1)-(2)). In this way, the POI to be displayed is efficientlytranslocated across the cytoplasmic membrane into the periplasm, thusenabling efficient incorporation of the POI into the phage particles.Preferred fusion proteins also comprise one of the filamentous phagecoat proteins or truncated versions of the coat proteins. In this case,the POI is anchored to the cytoplasmic membrane by the hydrophobicextension of the phage coat protein after translocation (FIG. 1) andbefore incorporation into the phage particle.

One particular example of a signal sequence targeting the SRP pathway isthe signal sequence of the E. coli protein DsbA (DsbAss). All otherelements of the exemplified pDST phagemids are derived from classicalphagemids such as the pAK100 series, which carry the signal sequences ofPeIB (PeIBss) or the PhoA (PhoAss) directing the polypeptides ofinterest to be displayed via the posttranslational Sec pathway (FIG. 2,(3)-(4)-(5)).

In one series of experiments, the display yields were compared betweenphage particles produced from pDST phagemids encoding PhoAss and pDSTphagemids encoding DsbAss. Phage particles were produced with standardprotocols as described below and purified by CsCl gradientcentrifugation. Western Blotting showed that for the display of asingle-chain Fv antibody the pDST phagemid produced phage particles thathave about the same display yields of the POI independent of the signalsequence used (FIG. 4A, scFv). In stark contrast however, all fourtested DARPins could, only be efficiently displayed when using theDsbAss containing pDST phagemids, and almost no protein displayed couldbe detected when using the PhoAss containing pDST phagemids (FIG. 4A,DARPins). Similarly, the DsbAss containing pDST phagemids resulted inconsiderably higher display yields in case of the polypeptides GCN4(FIG. 4A), lambda head protein D, TrxA and APH (FIG. 4B). Only slightlyhigher display yields were observed for proteins Taq polymerase, phageLambda protein phosphatase (λPP): and no displayed protein could bedetected in case of c-jun N-terminal kinase 2 (JNK2, FIG. 4B). Thisdemonstrates that the display yields on phage particles produced byusing DsbAss containing pDST phagemids are at least comparable to thoseproduced by classical phagemids using PhoAss, but show stronglyincreased display yields when displaying DARPins and other rapid foldedand thermodynamically stable proteins.

To quantify this difference, phage particles were used in phage ELISAexperiments. Phage particles displaying DARPins specifically binding theproteins APH and JNK2 were compared after production from either DsbAsscontaining pDST phagemids or PhoAss containing pDST phagemids. Based onthe detection of bound phage particles as quantified with an anti-M13antibody, an increased display yield of more than 100-fold was observed(FIG. 5).

To demonstrate that the higher display yields obtained by using DsbAssalso benefit selection experiments, two test mixtures containing threedifferent types of phage particles produced from phagemids encodingeither DsbAss or PhoAss were mixed in various dilutions. For both testmixtures phage particles displaying DARPins specifically binding theproteins APH and JNK2 were spiked at a 1:10⁷ dilution into phageparticles displaying unselected DARPins E3_(—)5 and E3_(—)19. These twotest mixtures were used as input libraries for standard phage displayselections on the target proteins APH and JNK2 (Table 2). Whereas theAPH and JNK2 specific phage particles could be enriched from the testmixtures produced from DsbAss-encoding phagemids around 1000-fold perselection cycle (already more than 10% of the tested clones werespecific for their target after only two cycles of selection), noenrichment from the test library produced from PhoAss containingphagemids could be observed even after five selection cycles (nospecific clones observed).

In another series of experiments, the display yields were compared byphage ELISA between phage particles produced from pDST phagemidsencoding PhoAss, PeIBss, DsbAss, TorTss, TolBss or SfmCss. Phageparticles displaying DARPins specifically binding the proteins APH andJNK2 were compared after production from individual pDST phagemids.Based on the detection of bound phage particles as quantified with ananti-M13 antibody, an increased display yield of up to 700-fold wasobserved with the SRP-dependent signal sequences (DsbAss, TorTss, TolBssor SfmCss) compared to the Sec-dependent signal sequence PhoAss. (FIG.6).

TABLE 1 Description of proteins displayed on the surface of filamentousbacteriophage Phagemid Protein a Abbr. PhoAss DsbAss Description b Ref.scFv_gpD scFv pDST24 pDST31 E1 - T245 of single-chain Fv c binding gpDcontaining a disulfide bond DARPin 3a 3a pDST22 pDST23 D13 - Q166 ofDARPin 3a d binding APH DARPin 2_3 pDST34 pDST37 D13 - Q133 of DARPin eJNK2_2_3 JNK2_2_3 binding JNK2 DARPin E3_5 E3_5 pDST30 pDST32 D13 - Q166of unselected f DARPin E3_5 DARPin E3_19 E3_19 pDST65 pDST66 D13 - Q166of unselected g DARPin E3_19 GCN4 GCN4 pDST39 pDST40 R249 - R281 of apeptide h derived from transcription factor GCN4 (E259-, S262P) pD□N2gpD pDST41 pDST42 T21 - V110 of the capsid i stabilizing protein ofbacteriophage λ JNK2□2 JNK2 pDST45 pDST46 S2 - R424 of mitogen-activatedj protein kinase JNK2 TrxA TrxA pDST47 pDST48 S1 - A108 of thioredoxin(TrxA k gene of E. coli) Stoffel fragment Taq pDST51 pDST52 S290 - E832of Taq DNA l Taq polymerase polymerase λ-phosphatase λPP pDST53 pDST54M1 - A221 of bacteriophage λ m Ser Thr protein phosphatase APH APHpDST55 pDST56 A2 - F264 of aminoglycoside n phosphotransferase (C19S,C156S, S194C) a scFv, single chain Fv antibody fragment; DARPin,designed ankyrin repeat protein; PhoAss, PhoA signal sequence; DsbAss,DsbA signal sequence b The first and last amino acids used are indicatedin single letter amino acid code, point mutations are mentioned c (SEQID NO: 2) d (SEQ ID NO: 3) e Binz, H. K. et al. loc. cit. f GenBankaccession number AAO25689 g GenBank accession number AAO25690 h (SEQ IDNO: 4) i Swiss-Prot accession number P03712 j Swiss-Prot accessionnumber P45984 k Swiss-Prot accession number P00274 l Swiss-Protaccession number P19821 m Swiss-Prot accession number P03772 nSwiss-Prot accession number P0A3Y5

TABLE 2 Enrichment of DARPins 3a and 2_3 presenting phage^(a) SignalCycle of panning sequence of (Positive colonies/amount of coloniestested)^(b) Antigen phagemid 1^(st) 2^(nd) 3^(rd) 4^(th) 5^(th) APHPhoAss 0/11 0/15  0/14 n.d. 0/9  DsbAss 0/14 4/16 14/14 n.d. n.d. JNK2PhoAss 0/14 0/16  0/14 n.d. 0/11 DsbAss 0/14 2/16 14/14 n.d. n.d.^(a)Input mixtures produced from phagemids encoding either PhoAss orDsbAss were produced as described in the Example. To a 1:1 mixture ofphage particles displaying the unselected DARPins E3_5 and E3_19, phageparticles displaying the target specific DARPins 3a or 2_3 were added ina 1:10⁷ dilution. ^(b)Colonies were screened by DNA sequencing

TABLE 3 Signal sequences Swiss SEQ Prot Acces- Abbrev. Source ID NO sionno. DsbAss E. coli thio-disulfide interchange 1 P0AEG4 protein DsbAPhoAss E. coli alkaline phosphatase PhoA 9 P00634 PelBss Erwiniacarotovora pectate lyase PelB 13 P11431 SfmCss E. coli chaperone proteinSfmC 14 P77249 TolBss E. coli protein TolB 15 P0A855 TorTss E. coliperimplasmic protein TorT 16 P38683

EXAMPLE

Materials

Chemicals were purchased from Fluka (Switzerland). Oligonucleotides werefrom Microsynth (Switzerland). Vent DNA polymerase, restriction enzymesand buffers were from New England Biolabs (USA) or Fermentas(Lithuania). Helper phage VCS M13 was from Stratagene (USA). All cloningand phage amplification was performed in E. coli XL1-Blue fromStratagene (USA).

Molecular Biology

Unless stated otherwise, all molecular biology methods were performedaccording to described protocols (Ausubel, F. M., Brent, R., Kingston,R. E., Moore, D. D., Sedman, J. G., Smith, J. A. and Stuhl, K. eds.,Current Protocols in Molecular Biology, New York: John Wiley and Sons,1999). Brief protocols are given below.

Phage Display Related Methods

Unless stated otherwise, all phage display related methods wereperformed according to described protocols (Clackson, T. and. Lowman, H.B. eds., Phage Display A Practical Approach, New York: Oxford UniversityPress, 2004; Barbas III, C. F., Burton, D. R., Scott, J. K. eds., PhageDisplay: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2001). Brief protocols are given below.

Cloning

A derivative of phagemid pAK100 (Krebber, A., Bomhauser, S., Burmester,J., Honegger, A., Willuda, J., Bosshard, H. R., and Plückthun, A., J.Immunol. Methods 201, 35-55, 1997) encoding the DARPin 3a was thestarting point for the cloning of the first phagemid of this study,called pDST23.

To replace the signal sequence of this pAK100 derivative, theoligonucleotides oDST4 (SEQ ID NO:5), oDST5 (SEQ ID NO:6), oDST6 (SEQ IDNO:7), and oDST8 (SEQ ID NO:8) were designed. These fouroligonucleotides encode the E. coli DsbA signal sequence. The E. coliDsbA protein can be found in the Swiss-Prot database (accession numberP24991). Its signal sequence is MKKIWLALAG LVLAFSASA (SEQ ID NO:1). Theoligonucleotides oDST4, oDST5, oDST6, and oDST8 were annealed andamplified with oligonucleotides oDST6 and oDST8 by PCR. The resultingDNA fragment encodes the DsbA signal sequence and is flanked by therestriction endonuclease sites XbaI and BamHI. This DNA fragment wasdigested with XbaI and BamHI and ligated into the similarly treated anddephosphorylated pAK100 derivative. The resulting phagemid pDST23 (FIG.3) was isolated and the correct sequence was verified by DNA sequencing.

To allow direct experimental comparison to phagemids encoding the signalsequence of PhoA (SEQ ID NO:9), a second phagemid called pDST22 wasgenerated. Again, four oligonucleotides—called oDST4p (SEQ ID NO:10),oDST5p (SEQ ID NO:11), oDST6 (SEQ ID NO:7), and oDST8p (SEQ IDNO:12)—were annealed and amplified with oDST6 and oDST8p by PCR. Theresulting DNA fragment encodes the PhoA signal sequence and is flankedby the restriction endonuclease sites XbaI and BamHI. This DNA fragmentwas digested with XbaI and BamHI and ligated into the similarly treatedand dephosphorylated pDST23. The resulting phagemid pDST22 was isolatedand the correct sequence was verified by DNA sequencing.

The other phagemids used in this study are listed in Table 1 and wereobtained as follows: The coding sequences of the proteins of interestwere PCR amplified using appropriate designed PCR primers and templateDNA, such as prepared cDNA or public available plasmid DNA. Thereby,either a BamHI or a BglII restriction sites was introduced 5-prime toeach of the coding sequences and two restriction sites (EcoRI and PstI)were introduced 3-prime to each of the coding sequences. These PCRfragments were digested either with BamHI or BglII and either EcoRI orPstI, and then ligated into the similarly treated and dephosphorylatedphagemids pDST23 or pDST22. The open reading frame of the expressioncassette for the fusion polypeptide comprising the cloned. PCR productwas maintained for all constructs, especially the correct reading framefor the C-terminal fusion to the C-terminal domain of phage protein III(CTp3) was maintained. The first and the last amino acids of the clonedproteins of interest are given in Table 1 as well as the reference oraccession number for either the GenBank orthe Swiss-Prot databases. Thecorrect sequence of all phagemids was verified by DNA sequencing.

For the designed ankyrin proteins (DARPins) 3a and 2_(—)3, phagemidsencoding the PelBss (pDST80 and pDST81, respectively), SfmCss (pDST86and pDST87, respectively), TolBss (pDST84 and pDST85, respectively) andTorTss (pDST88 and pDST89, respectively) were generated, using the samecloning strategy as described above for DsbAss and PhoAss.

Phage Production and Purification

5 ml 2xYT medium containing 1% glucose, 34 μg/ml chloramphenicol (cam)and 15 μg/ml tetracycline (tet) were inoculated with a single colony ofE. coli XL-1 Blue harboring the phagemid of interest and the cells weregrown overnight at 30° C. with shaking. Fresh 5 ml 2xYT mediumcontaining 1% glucose, 34 μg/ml cam and 15 μg/ml tet were inoculatedwith the overnight cultures at a ratio of 1:100 (OD₆₀₀=0.04) and grownat 37° C. to an OD₆₀₀ of 0.5 with shaking. The cultures were infectedwith VCS M13 helper phage at 4×10¹⁰ pfu (plaque forming units) per ml(multiplicity of infection ˜20) and the cells were incubated for 30 minat 37° C. without agitation and then for 30 min at 37° C. with shaking.The medium was changed by harvesting the cells by centrifugation (3500g, 24° C., 10 min) and resuspending the pellet in 50 ml of 2xYT mediumcontaining 34 μg/ml cam, 50 μg/ml kanamycin (kan) and 0.1 mMisopropyl-β-D-thiogalactoside (IPTG). After growth for 14 to 16 h at 30°C. with shaking, the cells were removed by centrifugation (5600 g, 4°C., 10 min). The culture supernatant was incubated on ice for 1 h withone-fourth volume of ice-cold PEG/NaCl solution (20% polyethyleneglycol(PEG) 6000, 2.5 M NaCl). The precipitated phage particles were thencollected by centrifugation at (5600 g, 4° C., 15 min) and redissolvedin 1 ml of TBS₁₅₀ (25 mM Tris/HCl, 150 mM NaCI, pH 7.5). Furtherpurification of the phage particles was done by CsCl gradientcentrifugation as follows. After addition of 1.6 g of CsCl, the volumewas adjusted to 4 ml with TBS₁₅₀. The CsCl solution was transferred intoa ½×1½ inch polyallomer tube (Beckmann, USA, No 358980) and centrifugedat 100000 r.p.m. for 4 h in a TLN-100 rotor (Beckman Instruments) at 4°C. After centrifugation the phage band was recovered. The phages weretransferred to ½×2 inch polycarbonate tubes (Beckmann, USA, No 349622),which were filled with TBS₁₅₀ to 3 ml. After centrifugation at 50000r.p.m. for 1 h in a TLA-100.3 rotor at 4° C., the pelleted phages wereredissolved in 3 ml TBS. After an additional centrifugation at 50,000r.p.m. for 1 h in a TLA-100.3 rotor at 4° C., the phages were dissolvedin 1 ml TBS. The total concentration of phage particles was quantifiedspectrophotometrically. The infective titer of the phage samples wasdetermined by titration on E. coli XL-1 Blue cells using 2xYT agarplates containing 1% glucose and 34 μg/ml cam. The colonies were countedafter overnight incubation at 37° C.

Phage Blots

5×10¹¹ phage particles, purified by CsCl gradient, were applied to 15%sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)under reducing conditions and transferred to a polyvinylidene fluoride(PVDF) Immobilon-P Transfer Membrane (Millipore, USA) byelectroblotting. The membrane was blocked with MTTBS₁₅₀ (TBS₁₅₀, 0.1%Tween 20, 5% skimmed milk) for 1 h at room temperature (RT) andincubated with a murine anti-pIII antibody (MoBiTec, Germany, No.PSKAN3) (1:1000 in MTTBS₁₅₀, 20 min at RT) as primary antibody, whichrecognizes the C-terminal domain of pIII. A F(ab′)₂ fragment goatanti-mouse IgG horseradish peroxidase conjugate (Pierce, USA, No. 31438)(1:10000 in MTTBS₁₅₀, 1 h at RD was used as secondary antibody. Theproteins were detected with ChemiGlow West substrate (Alpha Innotech,USA).

In a second experiment, the blocked membrane was incubated with murineanti-FLAG M1 antibody (Sigma, USA, No. F3040) (1:5000 in MTTBS₁₅₀, 1 hat RD as primary antibody. A goat anti-mouse IgG alkaline phosphataseconjugate (Sigma, USA, No. A3562) (1:10000 in MTTBS₁₅₀, 1 h at RT) wasused as secondary antibody. The proteins were detected with thesubstrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro bluetetrazolium (NBT) (Fluka, Switzerland).

Phage ELISA

Phage ELISAs were carried out to assay the amount of functionallydisplayed DARPins on M13 phage particles. Biotinylated APH and JNK2proteins (Binz et al., loc. cit.) were immobilized as follows:Neutravidin (66 nM, 100 μl/well; Socochim, Switzerland) in TBS₁₅₀ wasimmobilized on MaxiSorp plates (Nunc, Denmark, No. 442404) by overnightincubation at 4° C. The wells were blocked with 300 μl BTTBS₁₅₀ (TBS₁₅₀,0.1% Tween 20, 1% BSA) for 1 h at room temperature. Binding of thebiotinylated APH and JNK2 proteins (100 μl, 1 μM) in BTTBS₁₅₀ was donefor 1 h at 4° C.

Dilution series of phage particles in BTTBS₁₅₀ were added to the wellsand incubated at RT for 2 h. After washing the wells five times with 300μl TTBS₁₅₀ (TBS₁₅₀, 0.1% Tween 20) for 5 min, bound phage particles weredetected with anti-M13 horseradish peroxidase conjugate (AmershamPharmacia Biotech, UK, No. 27-9421-01) and soluble BM Blue POD substrate(Roche Diagnostics, Germany, No. 1484281).

Phage Panning

E3_(—)5, E3_(—)19, 3a and 2_(—)3 displaying phage particles wereproduced from either phagemids encoding the PhoA signal sequence(pDST30, pDST65, pDST22, pDST34) or from phagemids encoding the DsbAsignal sequence (pDST32, pDST66, pDST23, pDST37), respectively. Thesephage particles were used to prepare mixtures of phage particlesproduced from phagemids encoding either PhoAss or DsbAss. To a 1:1mixture of phage particles displaying the non-binding DARPins E3_(—)5and E3_(—)19, phage particles displaying the target-specific DARPins 3aor 2_(—)3 were added in a 1:10⁷ dilution. Biotinylated APH and JNK2proteins were coated as described for the phage ELISA. To each well 0.1ml of phage particle mixtures (10¹³ cfu/ml) were added to 0.1 mlBTTBS₁₅₀ and incubated for 2 h. After washing (3 times for the firstselection cycle, 4 times for the second cycle and 5 times for additionalcycles) with TTBS₁₅₀ and (3 times for the first selection cycle, 4 timesfor the second cycle and 5 times for additional cycles) with TBS₁₅₀, thephage particles were eluted by incubating for 15 min with 0.2 ml elutionbuffer (0.2 M glycine/HCl, pH 2.2) at about 22° C. followed by anelution for 30 min with 0.2 ml trypsin (10 mg/ml in TBS₁₅₀) at 37° C.The combined eluates (neutralized with 10 μl of 2 M Tris-base) were usedfor the infection of 4 ml of exponentially growing E. coli XL1-Blue.After 30 min at 37° C. without agitation and 30 min at 37° C. withshaking, the cells were spread on 2xYT agar plates containing 1% glucoseand 34 μg/ml cam and 15 μg/ml tet and grown overnight at 37° C. Thecells were washed from the plates with 2xYT containing 1% glucose, 15%glycerol, 34 μg/ml cam and 15 μg/ml tet and used for the phageproduction for the next cycle of panning. After each panning cycle, theidentity of 9 to 16 eluted phage particles was determined. This was doneby infection of E. coli with these phage particles and screening of thecolonies by PCR with clone-specific primers.

1-15. (canceled)
 16. A filamentous phage display method whereinpolypeptides of interest encoded by a DNA library are displayed on thephage particles and wherein the polypeptides of interest arecotranslationally translocated across the cytoplasmic membrane ofGram-negative bacteria.
 17. The method of claim 16 wherein thecotranslational translocation is accomplished based on the signalrecognition particle pathway.
 18. The method of claim 17 wherein thesignal sequence used for the cotranslational translocation based on thesignal recognition particle pathway of the polypeptide of interest to bedisplayed on the phage particle is a signal sequence promotingcotranslational translocation of TrxA.
 19. The method of claim 17wherein the signal sequence used for the cotranslational translocationbased on the signal recognition particle pathway of the polypeptides ofinterest to be displayed on the phage particle is selected from thegroup consisting of signal sequences of TorT, SfmC, FocC, CcmH, YraI,TolB, NikA, FlgI, and DsbA, and homologs thereof.
 20. The method ofclaim 19 wherein the signal sequence is selected from the groupconsisting of signal sequences of TorT, SfmC, TolB and DsbA.
 21. Themethod of claim 16 wherein the polypeptides of interest to be displayedon the phage particles are repeat proteins.
 22. The method of claim 16comprising the steps of (a) constructing a filamentous phage or phagemidvector containing an expression cassette for a fusion polypeptide thatpossesses an N-terminal signal sequence promoting cotranslationaltranslocation of the fusion polypeptide across the cytoplasmic membraneof Gram-negative bacteria; (b) constructing a combinatorial library ofphage or phagemid vectors by cloning of a DNA library encoding thepolypeptides of interests into the expression cassette of the vector ofstep (a); (c) transforming suitable Gram-negative bacteria with thelibrary of vectors of step (b); and (d) performing phage displayselection cycles to separate phage particles based on the properties ofthe displayed polypeptides of interest.
 23. A filamentous phage displaymethod wherein a polypeptide of interest displayed on the phage particleis cotranslationally translocated across the cytoplasmic membrane ofGram-negative bacteria using a signal sequence selected from the groupconsisting of signal sequences of TorT, SfmC, FocC, CcmH, YraI, TolB,NikA, FlgI, and DsbA, and homologs thereof.
 24. The method of claim 23wherein the signal sequence is selected from the group consisting ofsignal sequences of TorT, SfmC, TolB and DsbA.
 25. A phage or phagemidvector comprising a gene construct coding for a fusion proteincomprising the polypeptide of interest to be displayed on the phageparticle and a signal sequence selected from the group consisting ofsignal sequences of TorT, SfmC, FocC, CcmH, YraI, TolB, NikA, FlgI, andDsbA, and homologs thereof.
 26. The vector according to claim 25 whereinthe signal sequence is selected from the group consisting of signalsequences of TorT, SfmC, TolB, and DsbA.
 27. A library of phage orphagemid vectors comprising gene constructs coding for fusion proteinswherein each of the fusion proteins comprises a signal sequencepromoting cotranslational translocation of TrxA and a polypeptide ofinterest to be displayed on a phage particle wherein each polypeptide ofinterest is encoded by a member of a DNA library.
 28. The library ofvectors of claim 27 wherein the signal sequence is selected from thegroup consisting of signal sequences of TorT, SfmC, FocC, CcmH, YraI,TolB, NikA, FlgI, and DsbA, and homologs thereof.
 29. The library ofvectors of claim 27 wherein the signal sequence is selected from thegroup consisting of signal sequences of TorT, SfmC, TolB, and DsbA. 30.The library of vectors of claim 27 wherein the DNA library encoding thepolypeptides of interest is a DNA library encoding repeat proteins.